Bioscience Reports (2019) 39 BSR20190505 https://doi.org/10.1042/BSR20190505 Research Article BCYRN1 is correlated with progression and prognosis in gastric cancer Hongbing Zhai1 and Yanju Li2 1Department of Gastroenterology, Xianyang Central Hospital, Xianyang 712000, Shaanxi, China; 2Department of Pathology, Yanan University Affiliated Hospital, Yan’an 716000, Shaanxi, China Downloaded from http://portlandpress.com/bioscirep/article-pdf/39/11/BSR20190505/860279/bsr-2019-0505.pdf by guest on 23 September 2021 Correspondence: Yanju Li ([email protected]) Long non-coding RNA brain cytoplasmic RNA 1 (BCYRN1) has been found to play an im- portant role in tumorigenesis of a variety of tumors including gastric cancer (GC). However, the prognostic significance and molecular mechanism of BCYRN1 was still unknown inGC. In the present study, we found BCYRN1 expression was dramatically elevated in GC tis- sues and cell lines, and positively associated with tumor depth, lymph node metastasis and clinical stage in patients with GC. Moreover, univariate and multivariate Cox regression analyses demonstrated that high BCYRN1 expression was independent prognostic factor for overall survival in GC patients. In lncRNA-microRNA interactome database, we found that there were putative binding sites between BCYRN1 and miR-204-5p. Furthermore, we confirmed that down-regulation of BCYRN1 inhibited GC cell proliferation, migration andin- vasion through directly up-regulated miR-204-5p expression. In conclusion, BCYRN1 acts as a promising prognostic predictor in GC patients and regulated GC cell proliferation, cell cycle, migration and invasion through targeting miR-204-5p. Introduction Gastric cancer (GC) is the fourth prevalent cancer and the second leading cause of cancer deaths world- wide [1]. Although a decreasing incidence of GC was observed in many countries due to the changes in eating habits and environment, GC remains prevalent in East Asia, especially in China, Japan and Ko- rea [2]. Despite some advances in the diagnosis and treatment, the GC patients’ clinical outcome has not improved significantly at present [3]. In most countries, the 5-year overall survival rate for GC patients remains below 20% [4]. Therefore, the identification of mechanism of GC tumorigenesis can provide new insight to develop targeted therapy and improve the prognosis of GC patients. Long non-coding RNA brain cytoplasmic RNA 1 (BCYRN1), also known as BC200 RNA, is a 200-nucleotide non-coding RNA transcribed from human chromosome 2p21 between CALM2 and EP- CAM genes [5,6]. Originally, BCYRN1 was identified as a brain-specific non-coding RNA involved in neurodegenerative disease [7]. Subsequently, BCYRN1 was found to function as an oncogenic lncRNA in several types of human tumor [8]. In GC, BCYRN1 was increased in GC, and associated with TNM stage and tumor size [9]. In addition, BCYRN1 promoted GC cell proliferation, migration and invasion in vitro Received: 01 March 2019 [9]. However, the prognostic significance and molecular mechanism of BCYRN1 were still unknown in Revised: 04 September 2019 GC. Thus, we further explored the clinical and prognostic significance of BCYRN1 in GC patients through Accepted: 03 October 2019 analyzing the correlation between BCYRN1 expression and clinicopathologic features including overall survival. Moreover, we analyzed lncRNA-microRNA interactome database and found that there were pu- Accepted Manuscript online: 15 October 2019 tative binding sites between BCYRN1 and miR-204-5p. MiR-204-5p has been confirmed to function as Version of Record published: tumor suppressor in GC [10–16]. Therefore, we tried to explore the relationship between BCYRN1 and 15 November 2019 miR-204-5p in regulating GC cell proliferation, cell cycle, migration and invasion. © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 1 License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20190505 https://doi.org/10.1042/BSR20190505 Materials and methods Clinical specimens A total of fresh 127 GC tissue samples and 35 paired non-tumor tissue samples were collected from patients who underwent surgical resection or biopsy at Xianyang Central Hospital or Yanan University Affiliated Hospital. All clinical specimens were confirmed by pathologists, and stored under liquid nitrogen until use. None of the GC patients received any anti-tumor therapy prior to pathologic diagnosis. The study was approved by the Ethics Committee of Xianyang Central Hospital and Yanan University Affiliated Hospital. All experiments were conducted in compliance with government policies and the Helsinki Declaration. The informed consent was obtained from each patient. Quantitative real-time polymerase chain reaction Downloaded from http://portlandpress.com/bioscirep/article-pdf/39/11/BSR20190505/860279/bsr-2019-0505.pdf by guest on 23 September 2021 Total RNAs were extracted and isolated from tissues and cells by TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol. For detecting BCYRN1 expression, the PrimeScript RT Master Mix (Takara Biomedical Technology, Beijing, China) was used to synthesize complementary DNA, and One Step TB Green Prime- Script RT-PCR Kit (Takara Biomedical Technology, Beijing, China) was utilized to conduct quantitative real-time polymerase chain reaction (qRT-PCR) analysis in 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, U.S.A.). For detecting miR-204-5p expression, the Mir-X miRNA First Strand Synthesis Kit (Takara Biomedical Tech- nology, Beijing, China) was utilized to synthesize complementary DNA, and Mir-X miRNA qRT-PCR TB Green Kit (Takara Biomedical Technology, Beijing, China) was applied to perform qRT-PCR analysis. U6 and GAPDH served as endogenous controls. Cell culture and transfection TwoGCcelllinesBGC-823andAGS,andahumangastricepithelium cell line GES-1 (Carlsbad, CA, U.S.A.) were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, U.S.A.) in humidified air with 5% carbon dioxide and 37◦C. siRNA targeting BCYRN1 (si-BCYRN1), negative control siRNA (si-NC), miR-204-5p mimic, negative control mimic (mimics-NC), miR-204-5p inhibitor and negative control inhibitor (inhibitor-NC) were designed and synthe- sized by RiboBio Co., Ltd (Guangzhou, China). Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, U.S.A.) was used for cell transfection in accordance with the manufacturer’s protocol. Luciferase reporter assay The fragments of BCYRN1 containing miR-204-5p binding sites was amplified by PCR and cloned into the pmirGLO luciferase vectors (Promega, Madison, WI, U.S.A.), and named as wt-BCYRN1 and mut-BCYRN1, respectively. Then, GC cells were cotransfected with wt-BCYRN1/mut-BCYRN1 and miR-204-5p mimic/miR-204-5p inhibitor through Lipofectamine 3000 reagent. After 48 h, GC cells were collected and the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, U.S.A.) was used to detect the luciferase activity. Cell count kit-8 assay Cell viability was estimated by using Cell count kit-8 (CCK-8) assay at 24, 72, and 120 h after transfection. GC cells (2 × 104) were seeded in 96-well plates, and were added with 10 μl CCK-8 solution (Dojindo Molecular Technologies, Kumamoto, Japan) at the indicated time. Then, GC cells were incubated at 37◦C for 2 h, and the absorbance of the each well was detected using a microplate reader at a wavelength of 450 nm. Colony formation assay The transfected GC cells were seeded into six-well plates at density of 400 cells per well and cultured for 14 days in humidified air with 5% carbon dioxide and 37◦C. Then, the GC cells were fixed in methanol for 30 min and stained with Crystal Violet for 20 min. Colonies containing more than 50 cells were counted and compared. Cell-cycle analysis The cell cycle of GC cells was analyzed using flow cytometric analysis. The transfected GC cells were collected, and fixed with 70% ethanol at 4◦C overnight. Then cells were stained with 100 μl RNaseA and 400 μl Propidium Iodide for 40 min in the dark. Cell-cycle distribution was detected on the flow cytometry (FACScan, BD Biosciences, U.S.A.). 2 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20190505 https://doi.org/10.1042/BSR20190505 Transwell cell migration and invasion assays Transwell chambers were utilized to estimate cell migration and invasion abilities (Corning, Franklin Lakes, NJ, U.S.A.) with 8-μm pore size based on the manufacturer’s recommendations. For cell migration assay, 1 × 105 GC cells in 200 μl serum-free RPMI-1640 medium were added into the upper chamber, and 500 μl RPMI-1640 medium containing 15% FBS was added into the lower chamber. After incubation for 24 h, GC cells resting on the upper filter were removed by a cotton swab, and GC cells migrating to the lower chamber were fixed by ethanol and stained using Crystal Violet. For invasion assay, all steps were the same with that in migration assay except that the upper chambers were pre-coated with Matrigel (BD Biosciences, San Jose, CA, U.S.A.). The number of migrated or invaded cells at five random fields is counted under a microscope. Statistical analysis Downloaded from http://portlandpress.com/bioscirep/article-pdf/39/11/BSR20190505/860279/bsr-2019-0505.pdf by guest on 23 September 2021 All statistical data were analyzed using SPSS 22.0 statistical software (IBM Corp., Armonk, NY, U.S.A.). All experi- ments were independently repeated three times. The Student’s t test was utilized for statistical comparison between two groups. Survival curve was estimated by Kaplan–Meier method and log-rank test. The univariate and multivari- ate Cox proportional hazards’ models were used for evaluating prognostic variables in GC patients.